Instrumedics tape transfer/frozens/bone

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From:Gayle Callis <uvsgc@msu.oscs.montana.edu> (by way of histonet)
To:histonet <histonet@magicnet.net>
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We have been using the Instrumedics tape transfer system for frozens,
and will be using it for undecalcifid murine nasal turbinates.  I can
honestly admit that it is creme de la creme for cryosectioning difficult
frozen tissues!  Lungs do not have to be perfused with anything, but have
done this with OCT diluted 1:1 with PBS to expand the alveoli via perfusion
through the trachea (only 1 1/2 mls needed!), snap frozen with dry ice
isopentane slurry.

The Cryojane is the freezing component of this system, but for bone
frozen sections, Dodds et al, recommended using (demonstrated in a
workshop) bone dipped in a 4% PVA solution (Sigma 124,000-186,000)
and snap frozen in a dry ice/hexane slurry, we substituted this with
the 2 methylbutane), evaporating solvent off with specimen sitting on
dry ice.  Liquid Nitrogen temps can be too cold & can shatter bone, and
the Cryojane was too warm.  Mount the frozen bone onto a chuck with
either the PVA or OCT, cover specimen with OCT for sectioning, but with
the tape transfer, this probably is not necessary.  Since bone is so
hard, I prefer to mount it with 3% methylcellulose which gives rock hard
holding qualities to reduce chatter during sectioning.  Aldrich has this.

We are able to put the device in our particular cryostat without a permanent
installation, but some cryostats may not permit this, and the component
that flash polymerizes the polymer with UV light may need to be installed
and left in the cryostat.  A workable situation, in any case.

The slides are now available in 4X, 1X and 0.5X coatings with 4X
supposedly holding onto the tissue section better than 1X.  Personally
I like the 1X since it is less gooey to handle, but all in all, I
highly recommend the system.  A tidge more expensive, but I waste so
few slides since they are all usually good for IHC work, it breaks
about even in the long run.

The only thing I found to be annoying is the polymerized layer tends to
look bubbly with an aqueous mounting media, but solved that with Biomeda's
crystal mount or any equivalent (Shandon has one, Biogenex, etc) a liquid
coverslip barrier for AEC and solvent sensitive chromogens then you can
add a permanent coverslip.

I have sectioned mink skin that basically contains pure liquid form
lipid, worst stuff I ever saw! and the section was perfect, retaining
hair follicles usually lost with regular frozens since the cryostat
could not be set cold enough to solidify that yucky lipid content.
I takes just a few minutes to become proficient at using it, I taught
a grad student in 10 minutes!  Serial frozen sections are a snap

I have seen it demonstrated with larger bone sections but takes a good
sharp tungsten carbide knife, no matter what!  DDK is a good source
for these.

Now I hope Bernice sends me my commission for Christmas, the tape
transfer device was an excellent, timely investment and if the lab burns
down, I grab my purse, the Instrumedics device and run for the door! Is this
a hard sell/recommendation or what?!?

Happy thanksgiving to all!
Gayle Callis




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